DETECTION OF POLYMORPHISM IN THE TRYPANOSOMA-CRUZI TCP2-BETA GENE FAMILY BY SINGLE-STRAND CONFORMATIONAL-ANALYSIS (SSCA)

Single strand conformation analysis (SSCA) is a technique that has bee n used to detect point mutations. We explored its usefulness in the an alysis of four different members of the Trypanosoma cruzi TcP2 beta ge ne family and its suitability for detection of polymorphism in differe nt parasite strains. The availability of primers covering a 97-bp sequ ence at the 5' end of the genes allowed assessment of the effect of a single base substitution, while the analysis of a 321 bp long sequence permitted the evaluation of sequences differing in several bases. PCR products were analysed under four different electrophoretic condition s: with or without the addition of 10% glycerol in a 6% polyacrylamide gel run at room temperature or at 4 degrees C. Shifts in mobility wer e radically dependent on the migration condition. Both 97-bp and 321-b p amplicons were best resolved at 4 degrees C, without glycerol. Ampli fication products derived from total genomic DNA showed a pattern that resembled closely a combination of the products derived from the clon ed genes. The results herein demonstrate the usefulness of SSCA to dif ferentiate forms of a complex protozoan gene family, and to scan its p olymorphic nature. Furthermore, due to the remarkable sensitivity of t he technique it can generate genomic markers, such as Sequence Tagged Sites (STS), of great need in the T. cruzi genome project.